Dissecting and connecting the SigM stimulus and ESX-4 secretory response in mycobacteria - Mycobacteria utilize Type VII (ESX) secretion systems to export substrates across their complex cell envelope. Mycobacteria encode multiple paralogous ESX systems (M. tuberculosis encodes five ESX systems), which have evolved to perform non-redundant functions. Despite almost two decades of research, only ESX-1, -3 and -5 have been characterized and assigned functions in virulence, iron-uptake and cell wall integrity, respectively. The lack of a comprehensive understanding of the roles of ESX systems, their substrates and extracellular targets represents a significant gap in our understanding of mycobacterial biology. In this proposal, we will focus on ESX-4, which is considered the progenitor ESX secretion system because it is found in all mycobacteria. The absence of esx4 locus gene expression had confounded previous ESX-4 analyses and prevented its functional characterization. However, we demonstrated ESX-4 expression is triggered by direct cell-cell contact, which activates the extra-cytoplasmic sigma factor, SigM, to initiate transcription of the conserved core esx4 genes. Our data also show that SigM is dedicated to the regulation of an extended ESX-4-gene set, many of which are conserved in other mycobacterial species and are also controlled by SigM. The ability to directly control ESX-4 expression via SigM provides a unique opportunity to study the role of ESX-4 in both fast- and slow-growing mycobacteria. We have demonstrated ESX-4 is essential for conjugal DNA transfer in M. smegmatis and that ESX-4 induces changes in gross colony morphology and cell wall composition. In M. abscessus, ESX-4 plays a direct role in pathogenesis: it is required for intracellular survival in macrophages and amoebae, blocks phagosome acidification and promotes phagosomal rupture in infected macrophages. By extension, we hypothesize ESX-4 plays an important role in other pathogenic mycobacteria, mediated by ESX-4 secretion of effector molecules and remodeling of the cell wall. In this proposal, we will compare and contrast the ESX-4 systems of M. tuberculosis, M. marinum, M. abscessus and M. smegmatis, utilizing the ability to activate ESX-4 by SigM. Using a combination of biochemical, chemical biology, genetic, and molecular techniques we will define the SigM regulon and ESX-4 secretomes of each species. We anticipate identifying effector molecules that facilitate remodeling of the mycobacterial cell wall, as well as those that target other bacteria or the host macrophage. Together, this work will provide the first comprehensive analysis of ESX-4 and, importantly, significant new insights on the conserved and species-specific functions of this ancestral secretion apparatus.
